1. Field of the Invention
This invention relates to a signal processing method in autoradiography for determining base sequence of DNA or DNA fragment.
2. Description of the Prior Arts
Autoradiography has been known as a method for obtaining locational information on radioactively labeled substances distributed in at least one-dimensional direction to form distributed rows on a support medium.
For instance, the autoradiography comprises steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving the radioactively labeled biopolymers, derivatives thereof, cleavage products thereof or synthetic products thereof on a gel support (support medium) through a resolving process such as gel electrophoresis; placing the gel support and a high-speed X-ray film together in layers for a certain period of time to expose said film, developing said film; obtaining the locational information on the radioactively labeled substances from the developed film; and then performing the identification of the polymeric substances, determination of molecular weight thereof and isolation thereof based on the obtained locational information.
Recently, the autoradiography has been effectively used especially for determining the base sequence of a nucleic acid such as DNA (or DNA fragment, hereinafter "DNA" may be used to include both DNA and DNA fragment). The autoradiograhy is considered to be of a value for the structural determination of polymeric substances originating from organisms.
Sanger-Coulson method is known as a representative method for determining the base sequence of DNA. In this method, the base sequence of DNA is determined by ingeniously utilizing synthesis of DNA fragments with DNA synthesis enzyme, gel electrophoresis and autoradiography on the basis of such characteristic structure that DNA has a double helix structure consisting of two chain molecules, whose constitutional unit contains any one of four bases: adenine (A), guanine (G), cytosine (C) and thymine (T), the two chain molecules are stabilized through hydrogen bonding between these bases, and that the hydrogen bonding between each two constitutional base units comprises only two combinations, namely, G-C and A-T.
In the Sanger-Coulson method, there are a number of known procedures for synthesizing and sequencing DNA fragments complementary to DNA (hereinafter the sequenced fragments being referred to as DNA specimen). Basically, single-stranded DNA is used as template, which is incubated with a DNA synthesis enzyme (DNA polymerase) in the presence of mononucleoside triphosphates containing the above four kinds of bases, thus synthesizing (transcribing) DNA fragments of different but discrete lengths which are complementary to the DNA specimen. When part of the mononucleoside triphosphates are radioactively labeled, there can be obtained base-specifically synthesized DNA fragments (synthetic DNAs) labeled with a radioactive element.
A mixture of the synthetic DNAs is then resolved (developed) on a support medium by electrophoresis, and the support medium is autoradiographed to obtain an autoradiograph of a resolved pattern of the synthetic DNAs. The base sequence can be determined in order, starting from the end of the chain molecule according to the visualized autoradiograph. In this way, the sequence for all bases of the DNA specimen can be determined.
Sanger-Coulson method summarized above is described briefly in the following literature: "Reading the genetic information in the original language. A surprising method for sequencing the bases of DNA" written in Japanese by Kin-ichiro Miura, Modern Chemistry, September 1977, pp. 46-54 (Tokyo Kagaku Dozin Ltd., Japan).
In the autoradiography utilizing the conventional radiography, as described above, the visualization of an autoradiograph on a radiographic film is needed to obtain locational information on radioactively labeled substances. In fact, the investigators have determined the distribution of radioactively labeled substances in a sample by visually observing a visualized autoradiograph to obtain the locational information on the specific substances labeled with a radioactive element.
For instance, the DNA sequencing is visually determined by measuring the resolved positions of base-specific synthetic products labeled with a radioactive element and comparing them with each other. Accordingly, the autoradiography has unfavorable feature that it requires visual analysis, thereby needing a great amount of time and labor.
Further, the locational information obtained by the analysis of the autoradiograph varies depending on the investigators in charge because of inherent unreliability of visual observation, and the accuracy of the information is limited to a certain extent. Particularly, in such a case that only a small quantity of a sample has been employed, that the intensity of radiation energy emitted by the radioactively labeled substances has been low, or that the exposure has been done under unfavorable conditions, an autoradiographic image visualized on a radiographic film has reduced quality (in regard of sharpness, contrast, etc.) so that satisfactory information can not be obtained and the accuracy is low. These are drawbacks of the conventional autoradiography.
In order to improve the accuracy of the locational information, for instance, a visualized autoradiograph can been scanned by means of a device such as a scanning densitometer. However, such scanning process requires increased time and complicated procedures.
In addition to the above-described disadvantages, a sample and radiographic film is required to be placed together in layers for a long period of time to expose the film to radioactive substances contained in the sample so as to visualize the autoradiograph having the locational information on the radiographic film (for instance several days are required). Moreover, the exposure ought to be conducted at a low temperature (0.degree. C. to -90.degree. C.) to avoid appearance of the chemical fog of the photosensitive silver salt in the radiographic film which is caused by various substances contained in the sample. Thus, the exposure ought to be done under specific conditions. Moreover, since the photosensitive silver salt in a radiographic film has a drawback that it is also sensitive to physical impetus, or is apt to be physically fogged, careful and skillful handling is required. This feature further brings about another complexity in the autoradiographic procedure.
The radiographic film is also exposed to certain natural radioactive substances contained in the sample other than the radioactively labeled substances under analysis during the exposure for a long period of time. Accordingly, this exposure also lowers the quality of the obtained image, in addition to the chemical and physical fogs do.